Dietary Lactobacillus acidophilus and Mannan-Oligosaccharides Alter the Lipid Metabolism and Health Indices in Broiler Chickens

The effects of dietary Lactobacillus acidophilus (LBA) and mannan-oligosaccharide (MOS) supplementation on lipid metabolism and consequent lipid profile and health indices in broiler chicken were investigated in this study. Supplementation of 0.2% MOS along with either 106 or 107 LBA/g feed in broiler chicken downregulated hepatic expression of genes involved in lipogenesis, and upregulated expression of lipolytic genes. It caused decline of lipogenesis and increase of lipid oxidation which resulted in lower carcass fat content. None of the genes studied influenced fatty acid profile of chicken meat except the expression of stearoyl CoA (Δ9) desaturase-1 (SCD-1) whose upregulation increased monounsaturated fatty acid (MUFA) content at the cost of saturated fatty acid (SFA) content. The lipid metabolism indices of chicken meat such as ∆9 desaturase index (DI) increased in birds supplemented with 0.2% MOS along with either 106 or 107 CFU LBA/g feed, whereas no effect was observed on ∆5 + ∆6 DI. The supplementation of 0.2% MOS along with either 106 or 107 CFU LBA/g feed in birds improved the health indices of chicken meat due to upregulation of SCD-1 expression. The supplementation of 0.2% MOS along with either 106 or 107 CFU LBA/g feed in broiler chicken produced hypocholesterolemic and hypolipidemic effects with improved serum cardio-protective indices.


Introduction
The fat deposition in carcass of broiler chicken is the result of absorption, synthesis, and oxidation of lipids [1] which are determined by a balance between lipogenesis and lipolysis (β-oxidation) in mitochondria [2]. In birds, liver is the main seat of lipogenesis which is catalyzed by a series of linked enzymes [3,4]. The process of lipogenesis is initiated by acetyl-CoA carboxylase (ACC) followed by the repetitive reactions mediated by fatty acid synthase (FAS) [2]. However, in the process of lipogenesis, FAS requires NADPH, which is produced during the decarboxylation of malate to pyruvate and CO 2 by the action of malic enzyme (ME) in Krebs cycle [3,5]. The sterolregulatory elementbinding protein-1 (SREBP-1), a nuclear transcriptional factor, is highly expressed in liver compared to adipose tissue which induces the hepatic expression of lipogenic genes such as ACC, FAS, and ME [6,7]. It has been reported in various previous studies that mRNA expression of lipogenic enzymes such as FAS, ME, ACC, and stearoyl CoA (Δ9) desaturase 1 (SCD-1) is directly influenced by SREBP-1 and peroxisome proliferator activated receptor-α (PPARα) [3,7]. The SCD-1 is a rate-limiting enzyme in the synthesis of monounsaturated fatty acid synthesis (MUFA) in liver [8]. PPAR-α, a nuclear receptor predominantly expressed in liver, traffics fatty acids towards β-oxidation, and thus stimulates mitochondrial and peroxisomal fatty acid β-oxidation [9,10].
The lipogenesis carried out by ACC is controlled by AMP-activated protein kinase (AMPK) which acts by phosphorylation of ACC and inhibits its enzymatic activity [11]. However, lipid deposition in the tissues depends upon their transport by apolipoproteins [12], and apolipoprotein B100 (apoB100) is considered indispensable for the transport of lipids and their deposition in tissues [13]. It has been observed that abdominal fat accumulation in broiler chicken is associated with higher apoB48 and apoB100 containing lipoprotein particles as well as with upregulation of apoB gene expression [14]. Thus, the body lipid content is a result of a balanced interplay of all these genes involved in lipogenesis and lipolysis.
However, fat deposition in broiler chicken is a problem in poultry industry because it is an economic burden due to its negative impact on feed efficiency and has less acceptance among consumers who are conscious about nutritional quality of food [9,12]. The hepatic lipogenesis, lipid oxidation, and lipid transport in broiler chicken can be altered by nutritional modifications with the consequent alteration in lipid metabolism and tissue fat deposition [3,15]. The supplementation of dietary probiotics and prebiotics represents one such nutritional modification in broiler chicken. Probiotics are living microbial supplements with favorably improve gut microbiota to benefit host animal, whereas prebiotics are natural indigestible ingredients of food/feed that selectively stimulate the growth, composition, and activity of gut microbiota which results in improvement of host health and well-being [16]. The nutritional modifications do alter the expression of PPARs in broiler chicken hepatic tissues [17], and Lactobacillus fermentum has been reported to upregulate the PPAR-α pathway resulting in body fat reduction in mice [18]. Probiotics influence the course of digestive and metabolic processes in the body by altering the expression of genes involved in lipid and carbohydrate metabolism [19,20]. Probiotic supplementation have been shown to exert excellent lipid-lowering effects by depicting lower serum cholesterol and triglyceride levels in mice [20,21], whereas prebiotics positively improve lipid profiles by fostering the intestinal microbial population which produce short-chain fatty acids [16]. The prebiotics and probiotics have exhibited hypocholesterolemic and hypolipidemic effects of prebiotics and probiotics have been reported in other studies too [16,22,23].
Another aspect of lipid metabolism is its association with cardiovascular diseases whose prevalence has increased significantly due to changing food habits of people worldwide [24], and obesity is a multifactorial disease linked to the cardiovascular diseases [25]. In this study, broiler chicken was used as a model to assess the lipid metabolism in response to dietary Lactobacillus acidophilus (LBA) and mannan-oligosaccharides (MOS). Modern commercial broiler chicken strains are the result of intensive selection for rapid growth in poultry breeding, due to which they are hyperphagic and more prone to obesity. Thus, broiler chicken is a model of choice to understand the variations in lipid metabolism and consequent lipid profile and health indices in response to dietary LBA and MOS supplementation.

Supplements
For the present study, bacitracin methylene disalicylate (BMD) was purchased from ALPHARMA, Animal Health Division, New Jersey, USA, and had 44% bacitracin antibiotic activity. MOS was purchased from Kothari Fermentation & Biochem Ltd. New Delhi, India. The LBA (UBLA-34 MTCC 5401) of human fecal origin and characterized by whole genome sequencing was purchased from Unique Biotech Ltd. Hyderabad, India. The results of sequencing are deposited at DDBJ/ENA/GenBank under the accession number of RBHY00000000. As per the company descriptions, the LBA UBLA-34 is certified genetically safe as it does not contain any known virulence factors, antibiotic resistant genes, and plasmid. They are Gram-positive rods as cream to brown-colored powder and have water activity of less than one. The pathogens like E. coli, Salmonella, Staphylococcus, and Pseudomonas are absent in 10 g powder, and yeast-mould count are not more than 100 CFU/g (https ://www.uniqu ebiot ech.com/produ cts/food_and_bever ages/lacto bacil lus_acido philu s).

Birds, diets, management, and experimental design
A total of 288 1-day-old CARIBRO Vishal commercial broiler chicken of uniform body weight were procured from the institutional experimental hatchery. The birds were housed randomly in battery brooder cages with equal number of males and females for an experimental period of 42 days having eight birds in each battery providing a space of 0.75 ft 2 /bird. The birds were provided 24 h light for first 3 days followed by a decrease of 1 h per day till it reached 18 h light period which continued till 42nd day. For the thermal comfort of birds, starting cage temperature was 35 °C which was reduced by 2.8 °C every week. This study employed BMD, MOS, and LBA to formulate six dietary treatments, viz., T1 (negative control diet), T2 (positive control diet containing 20 mg BMD/kg diet), T3 (0.1% MOS + 10 6 CFU LBA/g feed), T4 (0.1% MOS + 10 7 CFU LBA/g feed), T5 (0.2% MOS + 10 6 CFU LBA/g feed), and T6 (0.2% MOS + 10 7 CFU LBA/g feed). Each treatment was assigned six battery cages (replicates) of birds (48 birds/treatment) randomly. The birds were provided ad libitum feed and fresh water for 42 days of feeding trial. The dietary supplementation levels of BMD, MOS, and LBA were standardized in a preliminary trial. The ingredients and nutrient composition of dietary treatments is given in Table S1. The formulated dietary treatments were isocaloric and iso-nitrogenous with similar fatty acid profile to prevent the potential confounding effects of variations in dietary energy, protein, and fatty acid profile on the results of the study.

Sample collection
Equal numbers of male and female birds were selected for sample collection to avoid sex as a possible confounding factor. At 21st and 42nd day of experimental trial, one bird was selected randomly from each replicate (6 birds/treatment) and sacrificed after 12 h of fasting with ad libitum drinking water for sample collection. The skinless fresh breast (two samples) and thigh samples (two samples) were collected from each sacrificed birds and stored at refrigeration temperature till fatty acid profile analysis. The liver samples were collected from each sacrificed bird for the study of hepatic expression of genes involved in lipid metabolism. The blood samples were collected in duplicate from each sacrificed birds in non-heparinized test tubes. The serum was harvested and stored at − 20 °C till further lipid profile analysis.

Hepatic expression of genes involved in lipid metabolism
The expression analysis of acetyl carboxylase (ACC), fatty acid synthase (FAS), malic enzyme (ME), apolipoprotein B100 (apoB100), sterolregulatory element-binding protein-1 (SREBP-1), stearoyl-CoA (Δ9) desaturase-1 (SCD-1), peroxisome proliferator activated receptor-α (PPAR-α), and AMP-activated protein kinaseα-1 (AMPKα-1) were carried out in liver using β-actin as housekeeping gene for normalization. Total RNA was extracted from each liver tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions, and treated with DNAse. The quantity as well as the purity of RNA samples was determined by measuring the absorbance at 260 and 280 nm by using spectrophotometer (Nanodrop 1000, Thermo Fisher Scientific, Singapore). RNA integrity was verified on 1.5% agarose gel by electrophoresis. The reverse transcription of RNA samples (1 µg) was carried out by using "Revert Aid™ First strand cDNA synthesis kit" (MBI Fermentas, Hanover, MD, USA) by following manufacturer's instructions. The quantity of cDNA samples was determined by measuring the absorbance at OD260/280 nm using Nanodrop (Nanodrop 1000, Thermo Fisher Scientific, Singapore). To check any possible genomic DNA contamination while RNA extraction a parallel negative control reaction with all the reaction components except RNA was also run. The resultant cDNA samples were stored at − 20 °C for further use.
The cDNAs were amplified by real-time quantitative PCR using IQ5 Cycler system (Bio-Rad, Hercules, CA, USA). Amplification was carried out in 20 µl reaction containing qPCR master mix of 1× SYBR GREEN dye (DyNAmo_HS; Finnzymes, Woburn, MA, USA), 0.2 µM concentration of 3′ and 5′ gene-specific primers (Table 1) and 2.5 µl of cDNA template. The qPCR conditions for 40 cycles were as follows: initial denaturation at 95 °C for 15 min, subsequent denaturation at 95 °C for 30 s, annealing at an optimized temperature ( Table 2) for 30 s, extension at 72 °C for 30 s. The gene-related primers and their optimized annealing temperature are listed in Table 1. All reactions were performed in nuclease-free 8 tube-strips with optically clear flat caps (Axygen Scientific Inc, Union City, CA, USA). Finally, following amplification process, dissociation curves were generated for all samples which were analyzed to determine the specificity of amplification. Results of amplification were expressed in terms of threshold cycle values (Ct), normalized against β-actin gene, and fold expressions were determined by ΔΔ CT method [26].

Chromatography and fatty acid profile
The fatty acid methyl esters (FAMEs) of meat sample were prepared as described by O'Fallon et al. [27] using C13:0 ME as internal standard. The prepared FAMEs were stored in GC vials and placed at − 20 °C till analysis. The Thermo Scientific Ceres 800 plus gas chromatograph (CP-6173) with 60 m × 0.25 mm × 0.20 mm capillary column (Varian) was used to determine the fatty acid composition of the FAMEs. The gas chromatograph was fitted with automatic sampler AI3000, integrator, and flame ionization detector. The initial oven temperature of 120 °C was held for 5 min and thereafter, increased to 240 °C at a rate of 2 °C/min which was held constant for 60 min. Nitrogen was used as carrier gas at a flow rate of 1 ml/min. The injector and detector of gas chromatograph were set at 260 °C with a split ratio of 30:1. Fatty acid standard purchased from Supelco, Bellefonte, PA, USA, contained 37 different FAMEs and 0.5 µl was injected into GC to get the standard peaks. The identification of different fatty acids was done by comparing their retention times with fatty acid methyl standards and were expressed as mg/g meat sample [24].

Lipid metabolism indices
The estimation of fat percentage (dry basis) of breast and thigh meat was done by refluxing of 2 g meat samples in petroleum ether for 5-6 h by using Soxhlet extraction apparatus [28]. The activities of desaturating enzymes which convert saturated fatty acids (SFAs) to monounsaturated fatty acids (MUFAs) are measured in terms of desaturase indices. These indices were calculated by comparing the percentage of product with that of precursor as follows [29]: The efficiency of enzyme activities in the synthesis of PUFAs from essential fatty acids (EFA), linoleic acid (LA), and ALA was measured in terms of Δ5 + Δ6 desaturase index, and enzyme activities involved in the conversion of myristic acid (C14:0) to palmitic acid (C16:0) and further to steric acid (C18:0) was measured in terms of thioesterase and elongase indices as follows [30]:

Health indices of meat
The health value of chicken meat was evaluated by considering the ratios of ω-6 to ω-3 PUFA, PUFA to SFA, MUFA to SFA, and UFA to SFA. Furthermore, the saturation index (S/P), atherogenic index (AI), thrombogenic index (TI) [31], desirable fatty acid (DFA) content, hypercholesterolemic fatty acids (HFAs), and hypocholesterolemic to hypercholesterolemic fatty acid ratio (h/H) [32] of chicken meat were calculated as follows:

Serum lipid chemistry and health-related indices
The serum triglyceride (TG), total cholesterol (TC), and HDL cholesterol estimation was done by using Span Diagnostic kits, Gujrat, India, according to the instructions of the manufacturer. The health indices were measured in terms of serum atherogenic indices such like cardiac risk ratio (CRR), atherogenic coefficient (AC), and atherogenic index of plasma (AIP) [33].

Statistical analysis
The experimental unit for data analysis was the sampled bird. Prior to analysis, all the data were tested for normality and homogeneity of variances with Shapiro-Wilk test and Levene's test, respectively. Following a completely randomized design, the data were analyzed by one-way ANOVA using general linear model procedure (IBM SPSS software-20). The group means were separated by Tukey post hoc analysis at a significance level of p < 0.05.

Hepatic gene expression at 21 days of age
The mRNA abundance of ACC, FAS, ME, SERBP-1, and apoB100 revealed a significant (p < 0.05) decreasing trend from treatment T1 to T6. The gene expression was higher in T1 and T2 followed by T3 and T4 compared to T5 and T6. The gene expression of PPAR-α, AMPKα-1, and SCD-1 showed a significant (p < 0.05) increasing trend from treatment T1 to T6 with lower expression in T1 and T2 followed by T3 and T4 compared to higher expression in T5 and T6. However, T1 and T2, T3 and T4, and T5 and T6 did not differ significantly from each other in terms of the expression of all these lipid metabolism genes ( Figs. 1 and 2).

Hepatic gene expression at 42 days of age
The trend of gene expression of ACC, FAS, ME, and apoB100 was similar to that at 21 days of age. The gene expression was significantly (p < 0.05) higher in T1 and T2 followed by T3 and T4 compared to T5 and T6. Again, the expression pattern of SCD-1 was similar to that at 21 days of age with lower expression in T1 and T2 followed by T3 and T4 compared to the higher expression in T5 and T6. However, the expression of SERBP-1, PPAR-α, and AMPKα-1 was not affected by dietary treatments (Figs. 3  and 4).

Fat percentage and fatty acid profile of chicken meat
The fat percentage and fatty acid profile of broiler chicken breast and thigh are given in Table 2 and Table 3, respectively. Significant and progressive decline was observed in fat (p < 0.01), palmitic acid (p < 0.01), stearic acid (p < 0.05), and SFA (p < 0.05) content, whereas progressive increase was observed in palmitoleic acid (p < 0.01), oleic acid (p < 0.05), and MUFA (p < 0.05) content of chicken meat from treatment T1 to T6. However, treatment T1 and T2, T3 and T4, and T5 and T6 did not differ significantly from each other. Other fatty acids remained statistically similar among the dietary treatments.

Lipid metabolism indices
In general, an increasing trend was observed in ∆9-DI (18), ∆9-DI (16), and total DI (p < 0.01), whereas significant decrease was observed in thioesterase index from treatment T1 to T6 in breast as well as thigh meat (Table 4). However, treatment T1 and T2, T3 and T4, and T5 and T6 did not differ significantly from each other. No treatment effect was observed on elongase index and ∆5 + ∆6 desaturase index.

Serum lipid chemistry and health-related indices
The serum lipid chemistry and health indices have shown significant dietary treatment effects ( Table 6). The serum TG (p < 0.01) and TC (p < 0.05) concentrations were higher in treatment T1 and T2 which did not differ significantly from T3, whereas lower concentrations were observed in T5 and T6 which were statistically similar to T4. The treatment T3 and T4 did not differ significantly from each other. The serum HDL cholesterol was higher (p < 0.01) in treatment T6 which was statistically similar to T5, whereas lower concentration was observed in T1 which did not differ significantly from T2 and T3. The serum health indices (CRR, AC, and AIP) were lower (p < 0.01) in treatment T6 which was similar to that in T5, whereas higher values were observed in T1 which did not differ significantly from T2. The treatment T3 and T4 resulted in intermediate values of health indices with T4 values significantly lower than that in T3.

Discussion
In modern times, new emerging food habits of people have led to a significant increase in the prevalence of cardiovascular diseases [24] and obesity is a multifactorial disease linked to the cardiovascular diseases [25]. Hepatic lipogenesis and lipid oxidation in broiler chicken is altered by dietary modifications [15]. Because of intense selection for faster growth in poultry breeding, modern broiler chicken strains are hyperphagic and more prone to obesity. Thus, the modern-day broiler chicken strains could be the models of choice to understand the variations in lipid metabolism and consequent lipid profile and health indices in response to dietary modifications. This study establishes the mechanistic understanding of lipid metabolism and health indices in chicken in response to dietary LBA and MOS supplementation. It was observed that the dietary supplementation of 0.2% MOS along with either 10 6 or 10 7 LBA/g feed resulted in lower fat content of chicken meat. This decline of fat content can be explained as a consequence of corresponding downregulation of genes involved in lipogenesis and upregulation of lipid oxidation genes. The downregulation of hepatic ACC, FAS, ME, SERB-1, and apoB100 expression, whereas upregulation of PPAR-α and AMPKα-1 was observed in birds supplemented with 0.2% MOS along with either 10 6 or 10 7 CFU LBA/g feed. The lipid synthesis is initiated by ACC, a complex multifunctional enzyme system, by catalyzing the carboxylation of acetyl-CoA to malonyl-CoA which is a rate-limiting step for both synthesis and elongation of fatty acid synthesis  Results are presented as means ± SEM with six birds per treatment. Mean values with different superscript letters differ significantly (p < 0.05). [34]. Following the action of ACC, repetitive reactions in the process of lipid synthesis are carried out sequentially by the action of multienzyme system FAS [2]. The main product of FAS reaction is palmitate (C16:0), but stearic acid (C18:0) and shorter fatty acids may also be produced [35]. The hepatic ME mRNA expression has been positively correlated with the rate of fatty acid synthesis and body fat content [5] because reducing equivalent NADPH required by FAS in the process of lipid synthesis is mainly provided by action of ME, which catalyzes the oxidative decarboxylation of malate to pyruvate and CO 2 with simultaneous generation of NADPH from NADP + [3,5]. Thus, in the present study, downregulation of ME expression was followed by downregulation of FAS expression with lower carcass fat content in synbiotic supplemented broiler chicken. On the other hand, SREBP-1 is a nuclear transcriptional factor which induces the expression of lipogenic genes such as ACC, FAS, and ME [6]. In the present study, it indicated that by downregulating the expression of SREBP-1, synbiotic supplementation downregulated the expression of main downstream lipogenic genes such as ACC, FAS, and ME resulting in lower carcass fat content.
Furthermore, apoB100 protein, synthesized in hepatocytes, is indispensable for the transport of lipids and their deposition in tissues [13]. The abdominal fat accumulation in broiler chicken has been associated with higher apoB48 and apoB100 containing lipoprotein particles as well as with upregulation of apoB gene expression [14]. The present study has shown a downregulation of apoB100 expression with corresponding decline of body fat content in synbiotic supplemented birds. This elucidates another possible mechanism by which the synbiotic supplementation has decreased the fat content of broiler chicken meat. Another set of genes pertaining to lipid oxidation evaluated in this study were PPAR-α and AMPKα-1. The PPAR-α, a nuclear receptor predominantly expressed in liver, stimulates mitochondrial and peroxisomal fatty acid β-oxidation by trafficking fatty acids towards β-oxidation [9,10]. Nutritional modifications   do alter the expression of PPARs in broiler hepatic tissues [17], and similar to the present study, Lactobacillus fermentum CQPC05 has been reported to upregulate the PPAR-α pathway resulting in body fat reduction in mice [18]. The activation of AMPKα by phosphorylation at Ser 79 inactivates AAC by its phosphorylation which in turn inhibits the production of malonyl-CoA used in lipogenesis. This mechanism fosters lipid oxidation on one hand and reduces lipid synthesis on the other hand [25,36]. Furthermore, AMPKα can directly decrease FAS gene transcription via regulating the gene expression of SREBP-1 in avian species [37]. However, age of birds do influence the rate of lipid synthesis to a considerable extent, due to which greater fat content is observed in carcasses of older chicken [38]. On similar lines, in the present study, it was observed that PPAR-α and AMPKα-1 were upregulated, and SERBP-1 was downregulated at 21 days of age but not at 42 days of age. It indicates that at 21 days of age, lipid oxidation process supersedes the lipogenesis process in broiler chicken due to which lower carcass fat content might have resulted compared to 42 days of age [12]. Stearoyl-CoA desaturase 1 (SCD-1), an endoplasmic enzyme, catalyzes the biosynthesis of MUFA from dietary or de novo synthesized SFA [9,39]. In the present study, upregulation of SCD-1 expression was observed in birds supplemented with 0.2% MOS along with either 10 6 or 10 7 CFU LBA/g feed, and in consequent to this, significant decrease in palmitic acid, stearic acid, and total SFA content of chicken meat was observed, whereas increase in palmitoleic acid, oleic acid, and total MUFA content was observed. From this study, it can be established that the genes studied do influence the fat content of chicken muscles but do not discriminate between different fatty acids except the SCD-1 gene which promotes MUFA synthesis at the cost of SFA.
The lipid metabolism indices evaluated in this study revealed that ∆9-DI (18), ∆9-DI (16), and total DI increased significantly in birds supplemented with 0.2% MOS along with either 10 6 or 10 7 CFU LBA/g feed because of SCD-1 upregulation. The upregulation of SCD-1 hastened the conversion of palmitic acid to palmitoleic acid which reduced the palmitic acid content resulting in to lower thioesterase index of chicken meat in this study. However, the conversion of stearic acid and palmitic acid by SCD-1 to their unsaturated counterparts seems to occur at same rate, thus exerts no significant effect on the elongase index of chicken meat. Furthermore, MOS and LBA supplementation did not have any significant effect on the ∆5 + ∆6 desaturase index which is corroborated by nonsignificant effect on the PUFA content of chicken meat. Notwithstanding the knowledge of authors, there is no research available pertaining to the study of lipid metabolism indices in response to dietary MOS and LBA supplementation in animals or birds to substantiate the results of this study. All the healthrelated indices of broiler chicken meat except ω-6:ω-3 PUFA ratio improved due to the supplementation of 0.2% MOS along with either 10 6 or 10 7 CFU LBA/g feed. All these health indices improved because of increase in MUFA content of meat at the cost of SFA content under the influence of SCD-1 upregulation. The ACC, FAS, ME, SERBP-1, PPAR-α, and AMPKα-1 expressions   analyzed in this study seem to have no effects on the health indices of chicken meat because they did not influence its fatty acid profile. No reports showing effects of synbiotic supplementation on the health indices of chicken meat are available.
The serum lipid profile can be a reliable indicator of the state of systemic lipid metabolism and deviation from normal values of main blood lipids such as TG, cholesterol, and HDL cholesterol are the most common markers of metabolic syndrome [40]. In the present study, lower serum TG and     [25]. In general, probiotics have been shown to exert excellent lipid-lowering effects by depicting lower serum cholesterol and TG levels in mice [20,21]. The probiotics hasten the enzymatic deconjugation of bile acids [41] and conversion of cholesterol to coprostanol in the intestines [42] and causes their elimination via feces. As a homeostatic response, more cholesterol is diverted towards synthesis of new bile acids which results in lowering of serum cholesterol. Further, by virtue of increasing gut viscosity and mucus layer thickness of intestines prebiotics inhibits cholesterol uptake and also enhances cholesterol breakdown in liver by which hypocholesterolemic effect is observed [23,43]. Previous studies employing dietary prebiotics and probiotics have reported hypocholesterolemic and hypolipidemic effects in animals [16,22,23]. Similar to the results of present study, Lactobacillus fermentum lowered serum total cholesterol and TG levels, whereas it increased HDL cholesterol in mice fed high-fat diet [18]. The improved cardioprotective indices like CRR, AC, and AIP observed in the present study may also be attributed to the enhanced absorption of micro-and macronutrients, including antioxidants in response to dietary synbiotic supplementation, which reduce postprandial lipids associated with oxidative damage and various cardiovascular pathologies [44,45].

Conclusion
In summary, the dietary supplementation of 0.2% MOS along with either 10 6 or 10 7 LBA/g feed in broiler chicken downregulates hepatic ACC, FAS, ME, SERB-1, and apoB100 expression, and upregulates the PPAR-α and AMPKα-1 expression. It causes decline of lipogenesis and increase of lipid oxidation which results in lower carcass fat content. The upregulation of PPAR-α and AMPKα-1 and downregulation of SERBP-1 occur at 21 days of age but not at 42 days of age. None of the genes studied influence the fatty acid profile of chicken meat except SCD-1 gene. The supplementation of birds with 0.2% MOS along with either 10 6 or 10 7 LBA/g feed upregulates SCD-1 expression resulting in increase of MUFA content at the cost of SFA content of chicken meat. The lipid metabolism indices of chicken meat such as∆9-DI (18), ∆9-DI (16), and total DI increase in birds supplemented with 0.2% MOS along with either 10 6 or 10 7 CFU LBA/g feed because of SCD-1 upregulation. The synbiotic supplementation exerts no effect on ∆5 + ∆6 DI which in turn has no effect on PUFA content and ω-6:ω-3 PUFA ratio of chicken meat. However, supplementation of 0.2% MOS along with either 10 6 or 10 7 CFU LBA/g feed in birds improve the health indices such as PUFA:SFA ratio, MUFA:SFA ratio, UFA:SFA ratio, saturation index, atherogenic index, thrombogenic index, DFA, HFA, and h/H ratio under the influence of SCD-1 expression. The supplementation of 0.2% MOS along with either 10 6 or 10 7 CFU LBA/g feed in broiler chicken produces hypocholesterolemic and hypolipidemic effects with improved serum cardioprotective indices. Significance: Probiotics are living microbial supplements which favorably improve gut microbiota to benefit host animal, whereas prebiotics are natural indigestible ingredients of food/feed that selectively stimulate the growth, composition, and activity of gut microbiota. The interaction of probiotics with animals has been extensively studied with most of the studies limited to assessment of gut microbial population, growth performance, and health status of animals. Though a number of studies reported hypocholesterolemic and hypolipidemic effects of prebiotics and probiotics, none of them provides in-depth understanding of the mechanisms behind these effects. None of the studies dealing with lipid profile and consequent health indices of chicken meat are available. This study provides a mechanistic understanding of lipid metabolism and consequent lipid profile and health indices of chicken meat in response to dietary Lactobacillus acidophilus and mannan-oligosaccharides.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of interest.
Ethical approval All applicable institutional guidelines for the care and use of animals were followed. The experimental procedures carried out in this study were approved by the Institutional Animal Ethics Committee (IEAC) following the guidelines of "Committee for the Purpose of